Project Summary/Abstract The circadian clock is necessary to synchronize biological processes with the environment. A properly timed clock relies on rapid creation and destruction of clock proteins. Despite this, few protein degradation mechanisms have been discovered that regulate the circadian clock of plants. This is likely due to genetic redundancy amongst the E3 ubiquitin ligases that control protein ubiquitylation. Arabidopsis has served as a powerful system for discovering molecular components of the circadian clock using live imaging for forward and reverse genetic screens. My laboratory has leveraged these advantages to perform two reverse genetic screens of E3 ubiquitin ligase families, overcoming traditional problems with genetic redundancy and identifying a host of new regulators of circadian clock function. This proposal describes the design and execution of this screen and the early functional characterization of newly discovered clock regulators. Functional characterization relies on our streamlined workflow that allows us to rapidly determine the E3 ubiquitin ligase substrate proteins, validate these potential substrates, and perform genetic and biochemical experiments demonstrating their role in clock function. The proposal then describes our two main laboratory goals moving forward: 1) completion of the proposed screens and 2) functional characterization of the newly discovered clock regulators. These studies will determine how protein degradation mechanisms can help clocks sense external signals, maintain a 24 hour rhythm, and connect to downstream rhythmic biological processes. The circadian clock regulates fundamental biological processes including photosynthesis, metabolism, defense, and growth. Thus, the work that we perform will have far-reaching impacts because: 1) it will provide the basic molecular building blocks that are necessary to generate a robust 24 hour clock in plants, 2) it will serve as a framework for similar studies in non-plant systems, and 3) it will provide a more comprehensive understanding of the post-translational mechanisms that overlay transcriptional feedback loops of clocks. Successful completion of this proposal will reveal how post-translational degradation mechanisms can survey cellular environments to control changes in transcriptional networks of circadian clocks and provide precise timing to clock-controlled biological processes. In the longer term, this work will increase our understanding of the regulation of critical biological processes in plants, but it will also translate to better understanding of clock function and clock-related diseases in humans.